控制水稻株高的脂质转移蛋白基因的克隆与分子机理研究

Plant height is not only a decisive factor that affects plant architecture but also an important agronomic trait that contributes to crop yield. So, elucidating the molecular mechanism for controlling plant height is significant for basic research of plant architecture patterning and for directing ideal plant architecture breeding. .We have isolated a new gene for controlling plant height by analyzing a dominant rice dwarf mutant due to somaclonal variation. Because it belongs to the non-specific lipid transfer protein (nsLTP) family, so we named it OsnsLTP. One base-pair deletion near the 3' terminal of the ORF caused frame shift in amino acid sequence, hence resulted in the dwarf phenotype. The mutant characters were recurred by transgenic experiment. Plnat nsLTPs are versatile, basic, soluble proteins that facilitate the transfer of fatty acids, phospholipids, glycolipids, and steroids between membranes. Gene members in this large family share highly conserved protein structures，usually contained 4-5 α helices forming an internal hydrophobic cavity stabilised by four intramolecular disulphide bonds. The hydrophobic cavity serves as the binding site for lipids. There are two closely-related types of nsLTPs, nsLTP1(9kDa) and nsLTP2(7kDa), which differ in protein sequence and biological properties. It was reported that nsLTPs may be involved in many important biological processes, including anther development, the formation of cutin layers on plant surfaces, plant systemic resistance signalling, and may play a key role in stress responses and the defense of plants against pathogens. Many nsLTP1 proteins have been characterized as allergens in humans. There are 52 nsLTP members in rice. Recently, it was reported that a nsLTP member OsC6 plays a crucial role in the development of lipidic orbicules and pollen exine during anther development in rice. Nevertheless, little information is known about the biological functions of a vast majority of these genes..It is well known that rice plant height is controlled mainly by GA, BR, PAT and SL related pathways, but the relation between nsLTPs and these pathways remains unknown. In view of the important roles of nsLTPs and yet the shortage knowledge of them, this project will focus on revealing the functions of OsnsLTP, clarifying its molecular mechanism for controlling plant height and give a preliminary investigation of the involving pathways. The researches including: 1) Revealing gene functions by analyzing mRNA expression patterns of the mutant and wild type, protein accumulation differences, gene knock-out and over-expression. 2) Elucidating the molecular mechanism by analyzing gene subcellular localization, the ability of lipid transfer, the role of conserved 8 cysteine motif, and screening interacting proteins. 3) Revealing related pathways through exogenous plant growth regulators treatments, transcriptome sequencing and iTRAQ analysis.

株高不仅是株型建成的决定因素，也是影响产量的重要农艺性状，揭示株高发育的分子机理对株型建成的基础研究和指导理想株型育种具有重要意义。我们从一个水稻显性矮化突变体克隆了1个控制株高的新基因OsnsLTP。植物非特异脂质转移蛋白（nsLTP）是结构保守、可转运多种脂分子的大家族，参与防御信号传导、角质形成、花粉发育等多种重要生命活动。目前对其生化结构研究较深入，但生物学功能和作用机制知之甚少。水稻有52个nsLTP成员，绝大多数的生物学功能尚未知。水稻株高主要由GA、BR、PAT和SL等相关途径基因控制，nsLTP与这些途径的关系不明。本研究的目标和内容是：1）通过分析突变体和野生型目标基因表达模式和蛋白积累差异、基因功能敲除和过表达转化以阐明该基因功能；2）通过亚细胞定位、互作因子筛选、脂质结合能力分析及保守结构域突变分析等揭示其作用机制；3）通过激素处理和表达谱分析初步了解调控途径。

前期研究中获得一个光周期-温度敏感型水稻矮化突变体Photoperiod-Thermo-sensitive Dwarf 1(PTD1)，其在长日低温表现严重矮化，短日高温恢复株高。我们克隆了该基因PTD1。其野生型基因ptd1预测编码非特异脂质转移蛋白（nsLTP）。nsLTP是结构保守、可转运多种脂分子的大家族，参与多种重要生命活动。水稻有52个nsLTP 成员，绝大多数的生物学功能尚未知。研究PTD1矮化突变体不仅有助于了解nsLTP家族成员的功能，还将揭示一种全新的株高控制机制，并为理想株型育种提供新途径。本研究的目标和内容：1）通过分析突变体和野生型目标基因表达模式、基因功能敲除和过表达转化以阐明基因功能；2）通过亚细胞定位、蛋白积累差异、脂质结合能力分析及保守结构域突变分析等揭示其作用机制；3）通过激素处理、互作因子筛选和表达谱分析初步了解调控途径。获得以下结果和结论： 1）PTD1控制株高是显性获得性功能：突变体不论杂合、纯合均矮化；野生型中转入PTD1即表现矮化。2）PTD1控制株高是一种新的机制，矮化效应依赖于ptd1保守半胱氨酸基序的破坏及合适的C端结构：PTD1对外施GA/BR的反应与野生型一致，外施GA/BR不能使其恢复株高，因而不是GA/BR生物合成及感知缺陷型矮化突变体；PTD1因突变移码丧失最末2个保守Cys，并形成移码C端；PTD1与ptd1 mRNA表达水平没有显著差异，而PTD1蛋白的脂质分子结合能力比ptd1显著下降；在纯合突变体中，CRISPR/Cas9敲除PTD1的功能后株高恢复正常；野生型植株转入点突变了特定Cys的ptd1可导致矮化，而转入同样丧失最末2个Cys、但不带C端尾巴的ptd1却表现正常；3）矮化突变体对光周期的响应可能是PTD1的互作因子或其下游因子的效应：通过长、短日处理PTD1突变体分别获得了矮化、正常株高的PTD1植株，野生型长、短日处理株高均正常，但二者在长、短日条件均可检测到蛋白积累且无差异。4）PTD1可能涉及蛋白降解途径：通过酵母双杂交实验筛选到一个编码泛素结合酶E2的互作因子，其与ptd1的C端互作，而与PTD1的C端不互作；5）科学意义和应用：揭示了一种突变型nsLTP蛋白控制株高的新机制，发明了一种通过点突变nsLTP基因实现株高控制的分子育种新方法。